A role for ppGpp in transcription-coupled DNA repair. Nucleotide excision repair (NER) of bulky DNA lesions occurs first through UvrAB recognizing and binding to the lesion, which leads to recruiting UvrCD repair functions. Alternatively, template strand lesions can lead to RNA polymerase arrest and backtracking of RNA transcripts, which obscures the lesion and prevents repair. Efficient transcription-coupled repair (TCR) requires exposing the lesion by either pushing or pulling the stalled polymerase. The Mfd protein binds polymerase and DNA downstream, then pushes polymerase forward to uncover the lesion. Pulling of polymerase back from the lesion involves binding of NusA to the polymerase -flap together with UvrD bound to both polymerase and sequences upstream of the single strand/double strand DNA fork junction. Dimerization of the UvrD helicase together with bound NusA then pulls the complex back to expose the DNA lesion. This allows recruitment of UvrAB to the now naked lesion to initiate repair. Modest elevations of basal ppGpp levels are found to be induced by the damage itself, which function to enhance dimerization of UvrD necessary for its binding. Relations between ppGpp, DksA and GreA/B have also been explored. When greAB are deleted, the otherwise sensitive ppGpp0 cells became damage resistant. Also deleting dksA sensitizes wild type cells to DNA damage while overexpressing DksA confers resistance. This infers that ppGpp facilitates backtracking, which was verified by in vitro transcription with purified components. Furthermore, exonuclease III mapping of RNAP assembled into Kashlev elongation complexes showed the expected ppGpp-dependent position shifts as well as displaying consistent in vivo footprint changes. Finally, the effects of ppGpp were mimicked by the RNA polymerase mutant (RpoB*35), previously found by the Lloyd group to phenocopy several regulatory effects of ppGpp. The suggested role of ppGpp is to loosen the DNA clamp. The ability of ppGpp to inhibit rRNA synthesis and thereby alter the coupling of transcription and translation can be viewed as additional features of the stringent response that ensure genomic integrity. The role of ppGpp in DNA synthesis initiation. Many stress conditions can provoke elevation of ppGpp but only two are well understood. The first involves sensing any amino acid metabolic deficiencies that interfere with aminoacylation of cognate specified tRNA; uncharged tRNA activates the strong RelA ppGpp synthetase bound to ribosomes. The other stress sensed is limited lipid synthesis, which through binding of nonacylated ACP (acyl carrier protein) to SpoT protein activates its weak synthetase and inhibits its strong hydrolase. We are ignorant as to how other stress responses control the two ppGpp synthetases and single hydrolase. Our genetic approach to this question is to construct a series of strains that allow each of the three activities to be compared during a given stress, in this case glucose starvation. This led to classical diauxic shift experiments that revealed a major effect when relA synthetase was absent; the time required to adjust during shifts to a new sugar was markedly slowed compared to wild type. The absence of the weak SpoT synthetase gave weak effects, as if ppGpp mediates adjustment to diauxic shifts from glucose to other carbon sources and more ppGpp functions better. As expected from its known specificity, the relA-dependent defect could be overcome by adding all 20 AA even though the shifts involved glucose starvation. We were surprised to learn that adding succinate provided the missing AA during glucose shifts. These were found to be arg, lys and met. The question then turned to what metabolites related to succinate were regulated by ppGpp. One clue is that aceE and ppGpp0 are synthetic lethals. Another is that ackA, but not pta, abolish the prolonged diauxic lags of relA strains. Abolishing the lag also occurred in the presence of succinate, as if AA starvation-induced ppGpp is necessary for the ackA deletion to facilitate the accumulation of acetyl phosphate (AcP). At about the same time we learned that the Swaleska lab had found that the ts growth of a famous dnaA46 allele was suppressed by aceE, ackA or pta, again implicating AcP. The ATP-dependent oligomerization of DnaA at its binding site repeat sequences within the origin (ori) region is the first step known in the complex multistep process of initiation of chromosomal DNA replication. A recent Nature Report (Zhang et al. 2016 PMID: 27484197) convincingly showed that a specific acetylation of the K178 lysine found in the Walker ATP binding motif reversibly inhibits DnaA oligomerization at origins. Classically, qPCR ori/ter ratios of ori DNA to DNA in the terminator region (ter) are measures of initiation. With mutants that vary basal ppGpp levels, AcP abundance was found to increase over a nearly 10-fold range as basal ppGpp levels increase. This led us to suspect that increased ppGpp might lead to nonenzymatic AcP-mediated acetylation of the key DnaA K178 residue, which would inhibit its ability to stimulate origin activity. However, this guess was disproven, since when AcP was abolished by introducing ackA or pta double deletions, ori/ter ratios remained regulated. This is taken to mean that while ppGpp is a regulator it does not work through AcP acetylation. Indeed, inhibition of DNA initiation at very slow growth rates was accompanied by increased ppGpp levels and by ori/ter ratios approaching unity. Conversely, ppGpp0 strains completely lacking ppGpp were found to have ori/ter ratios of 3-4 even during very slow growth as if regulation was abolished. This suggests the absence of ppGpp can override the effect of slow growth, ie ppGpp is necessary for regulation. Studies with strains whose elevated levels of ppGpp were achieved by constitutive mutations rather than because of slow growth were found to have lowered ori/ter ratios, as if a simple increase of ppGpp is sufficient to give regulation of initiation of DNA synthesis. The logical statement of this behavior is that ppGpp is both necessary and sufficient for regulation. This does not mean ppGpp is the only regulator nor does it reveal the mechanistic details. The initiation process is complex many components defined by a variety of incisive experiments of others. We also could demonstrate wild type regulation in ppGpp0 strains that harbor RNA polymerase mutants that mimic the effects ppGpp has on gene expression. This implies that ppGpp effects regulation indirectly, by altering on gene expression. Future studies will be aimed at dissecting out this mechanism in more detail.